CHARACTERIZATION OF A MAMMALIAN REPAIR GENE

哺乳动物修复基因的特征

• 批准号: 6497565
• 负责人: THOMAS Dominic STAMATO
• 金额: $ 28.52万
• 依托单位: LANKENAU INSTITUTE FOR MEDICAL RESEARCH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-15 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6497565
• 关键词: CHO cells; DNA damage; DNA directed DNA polymerase; DNA repair; DNA replication; complementary DNA; enzyme activity; gene complementation; gene mutation; gene targeting; glutathione transferase; immunoprecipitation; monoclonal antibody; transcription factor

DESCRIPTION:Although it is known that normal mammalian cells employ mutagenic replicative bypass as a tolerance mechanism for certain DNA lesions, much remains to be learned about this pathway of DNA repair/damage tolerance at the molecular level. A CHO mutant, called CHO-UV1, showing defective mutagenic bypass repair has been isolated. UV1 is hypersensitive to killing by various DNA damaging agents including alkylating drugs, mitomycin C and UV, but not ionizing radiation. UV1 has normal nucleotide excision repair but is defective in bypass repair and post replication recovery (PRR). It is less mutable than wild type CHO implying that the normal UV1 "gene" function is mutagenic. The phenotype of UV1 can be complemented by transfecting human genomic DNA into UV1 or by fusing UV1 with normal human cells, suggesting that human cells possess an equivalent of the gene mutated in UV1. A human cDNA encoding a truncated form of the ALY transcriptional coactivator has been found to complement the MMC killing hypersensitivity of UV1. Antisense-ALY, on the other hand, increases CHO's sensitivity to MMC. A fully MMC-resistant human-UV1 hybrid has been found to contain a fragment of human chromosome 11p with the ALY gene in it. The preliminary evidence thus implicates ALY as a candidate for the UV1 "gene" and a role for ALY in bypass repair and PRR. Studies are proposed to: (1) Investigate in detail the role of ALY in the UV1 PRR repair pathway, (2) examine the possibility that another gene can complement the UV1-PRR pathway and further characterize the phenotype of the mutant, and (3) examine the biochemical role of ALY (and/or other complementing genes) in the mutagenic PRR process.

描述：尽管已知正常哺乳动物细胞采用诱变 复制旁路作为某些DNA病变的耐受性机制，很多 关于DNA修复/损伤耐受性的这种途径还有待了解 分子水平。一个称为cho-uv1的cho突变体，显示出缺陷的诱变 旁路维修已隔离。 UV1对杀死各种 DNA损伤剂在内，包括烷基化药物，丝裂霉素C和UV，但不 电离辐射。 UV1具有正常的核苷酸切除修复，但有缺陷 在旁路维修和复制后恢复（PRR）中。它比不可能 野生型CHO表明正常的UV1“基因”功能是诱变的。这 UV1的表型可以通过将人基因组DNA转染到UV1来补充 或通过将UV1与正常的人类细胞融合，表明人类细胞拥有 等效于UV1中突变的基因。编码截短的人cDNA 已发现ALY转录共激活因子的形式与 MMC杀死UV1的超敏反应。另一方面 提高了Cho对MMC的敏感性。完全耐MMC的人类UV1混合动力车具有 发现与Aly Gene中的Aly基因中包含一个人类染色体的片段 它。因此，初步证据暗示Aly是UV1的候选人 “基因”和Aly在旁路修复和PRR中的作用。 提出了研究：（1）详细研究ALY在UV1中的作用 PRR修复途径，（2）检查另一个基因可以 补充UV1-PRR途径，并进一步表征 突变体和（3）检查Aly的生化作用（和/或其他补充 基因）在诱变PRR过程中。